A series of whole animal studies have clearly demonstrated selective brain neurodegeneration in a binge-type alcohol rodent model. Pyramidal cells of layer III of the entorhinal cortex (ENT) and hippocampal (HIPP) dentate gyrus granule cells are particularly vulnerable to the cytotoxic consequences of binge-type alcohol exposure. The main purpose of this project was to establish an experimental model to investigate alcohol-induced neurodegeneration in vitro that would complement our in vivo studies. Organotypic rat brain slices provide a culture system that preserves the main morphological and neurochemical features of brain tissue.Furthermore, organotypic slice cultures retain their native circuitry, cell types and synaptic density with conservation of main afferents.Ongoing studies have revealed that the ENT-HIPP explants are optimal for study at 2-3 weeks post harvest when slices are obtained from 8-day old neonates. The ENT-HIPP explants were treated with ethanol (0.2-0.6%) over 6 days using several different alcohol exposure paradigms. Alcohol-induced neuronal injury and/or death were assessed by the extent of uptake of propidium iodide(cytotoxic marker) and the amount of lactate dehydrogenase(LDH), another cytotoxic marker, in the explant medium. Treatment of ENT-HIPP slices with 0.4% alcohol for 6 days in the absence of antioxidants (glutathione, vitamin E,catalase and superoxide dismutase) caused a consistent, significant increase in medium LDH which paralleled propidium iodide uptake. Inclusion of antioxidants was cytoprotective.These results suggest that oxidative stress is an important mediator of alcohol-induced brain damage. Additional studies are needed to determine whether or not alcohol treatment of organotypic brain cultures will provide a useful model for our whole animal studies.